Methods for natural product optimization

ABSTRACT

Methods and compositions for natural product optimization are provided. In particular, methods and compositions for selecting bacterial strains (e.g., predatory bacteria such as myxobacteria) which produce a desired compound (e.g., antibiotic, antifungal, or anticancer agent) are provided.

The present application is §371 application of PCT/US2009/049562, filedJul. 2, 2009, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/077,659, filed on Jul. 2, 2008.The entire disclosure of each of the foregoing applications isincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to drug optimization. More specifically,the invention relates to methods of selecting bacterial strains,particularly myxobacteria strains and other predatory gliding bacteria,which produce a desired antibiotic, antifungal, or anticancer compound.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated by referenceherein as though set forth in full.

The discovery and implementation of antibiotic drugs nearly 70 years agorevolutionized human medicine. Just a short time ago infectious diseaseswere the number one killer of Americans. Largely because of antibioticdevelopment, infectious disease is no longer the leading cause of deathin the U.S. and the average life expectancy has increased by nearly 30years. The spectacular advancement of antibiotics from the mid-1940's to1961 lead the U.S. Surgeon General to proclaim before Congress in 1969that it is “time to close the book on infectious disease as a majorhealth threat.” In the decades that followed, it was found that heavyantibiotic use had resulted in an alarming increase in bacterialresistance (Talbot et al. (2006) Clin. Infect. Dis., 42:657-668). Inparallel to the increase in resistance, major pharmaceutical companiesabandoned antibiotic discovery in favor of life style and chronicdisease drugs that offer larger profit margins. Consequently, only twonovel classes of antibiotics have been introduced in the past 35 years.In 2004, the Infectious Disease Society of America released a reportthat a public health crisis is brewing as antibiotic research stagnates.Statics support this claim: (i) 70% of hospital acquired infection areresistant to at least one major class of antibiotics; (ii) 2 millionpatients acquire hospital bacterial infections per year resultingin >90,000 deaths, a number that has increase 6-fold since 1992, (iii)drug resistant infections cost the U.S. economy $5 billion annually, and(iv) a number of pathogens, such as methicillin resistanceStaphylococcus aureus (MRSA) which represents 70% of S. aureus hospitalacquired infections, are multidrug resistant and difficult to treat withexisting antibiotics. Because of these factors there is an urgent needto discover and develop new antibiotics that work by novel mechanism(Talbot et al. (2006) Clin. Infect. Dis., 42:657-668; Norrby et al.(2005) Lancet Infect. Dis., 5:115-119; Payne, D. J. (2008) Science321:1644-1645).

Natural products (NPs), which are by far the leading source ofantibiotics, are developmentally hindered by low fermentation yields andstructural complexity that make synthesis and optimization difficult(Walsh, C. (2003) Nat. Rev. Microbiol., 1:65-70; Baltz, R. (2007)Microbe 2:125-131; Demain et al. (2008) Prog. Drug Res., 65:251,253-289). For these and other reasons, pharmaceutical companies havelargely abandoned NPs and developing new antibiotics (Walsh, C. (2003)Nat. Rev. Microbiol., 1:65-70; Baltz, R. (2007) Microbe 2:125-131;Baltz, R. H. (2008) Curr. Opin. Pharmacol., 8:557-563; Overbye et al.(2005) Drug Discov. Today 10:45-52; Nathan et al. (2005) Nat. Rev. DrugDiscov., 4:887-891). Even with the advent of robotics andhigh-throughput technology, laborious screens used by pharmaceuticalindustries are still limited in their ability to process tens tohundreds of thousands of mutants (Demain et al. (2008) Prog. Drug Res.,65:251, 253-289; Baltz, R. H. (2001) Antonie Van Leeuwenhoek,79:251-259). To date, predation has not been utilized for naturalproduct optimization, in part, because the major natural productproducers (actinomycetes) are not predatory bacteria.

SUMMARY OF THE INVENTION

In accordance with one aspect of the instant invention, methods forscreening for a natural product producing strain of a predatorymicroorganism are provided. In one embodiment, the method comprises:obtaining a predatory microorganism, culturing the predatorymicroorganism under conditions wherein they kill and consume prey cells,and isolating a predatory microorganism(s) which grows on the prey cells(e.g., the predatory organism which grows best). In one embodiment, thepredatory microorganisms are cultured under conditions wherein preycells are the only nutrient source. In a particular embodiment, thepredatory microorganism cannot utilize (prey) on the prey cells (i.e.,prior to the selective pressure of the culturing step).

In yet another embodiment, the method comprises: obtaining a predatorymicroorganism, mutagenizing the predatory microorganism (e.g.,contacting the predatory microorganism with a chemical or physicalmutagen or directed nucleic acid mutagenesis (e.g., ta1 knockout),culturing the mutagenized predatory microorganism under conditionswherein they kill and consume prey cells (e.g., wherein the onlynutrient source is prey cells), and isolating a mutagenized predatorymicroorganism(s) which grows better on the prey cells than theunmutagenized predatory microorganism. In a particular embodiment, theunmutated or wild-type predatory microorganism cannot utilize (prey) onthe prey cells.

The predatory microorganism of the instant methods may be a myxobacteriaor other predatory gliding bacteria. Furthermore, the prey cell may be abacteria, fungus, parasite, mammalian cell, or cancer cell. The methodsmay also further comprise isolating the natural product(s) produced bythe isolated mutagenized predatory microorganism. In another embodiment,the natural product may be an antimicrobial compound, an antibioticcompound, an antifungal compound, an anticancer compound, or anantiparasitic compound. In another embodiment, the natural product issecreted by the predatory microorganism.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1J provide the structures of secondary metabolites produced bygliding bacteria and have antibacterial activity. All compounds areproduced by myxobacteria, except TAN-1057, which is produced byFlexibacter. The compounds are: A) antibiotic TA, B) ripostatin, C)corallopyronin, D) myxopyronin, E) sorangicin A, F) tartrolon, G)sorangiolid, H) angiolam, I) althiomycin, and J) TAN-1057.

FIGS. 2A and 2B are graphs providing the fraction of survival of certainGram-negative prey bacteria and Gram-positive prey bacteria,respectively, in the presence of Myxococcus xanthus (DK1622). Underthese starvation conditions prey cells are fully viable (>three days).Bacteria were grown in rich media, concentrated, and then mixed inbuffer (TPM) and spotted onto starvation agar plates. At various times,bacteria spots were collected and viable prey colony forming units (cfu)were measured on LB plates where M. xanthus growth is inhibited (highsalt). FIG. 2C is a graph demonstrating that antibiotic TA biosynthesisis required for predation. Isogenic strains of Myxococcus xanthus werecompared for their predation activity on ½ casitone-Tris (CTT) plates.TA−=DK1622 with an insertion mutation on the TA1 gene.

FIGS. 3A-3D demonstrate TA activity. DK1622 (WT) culture extract (FIG.3A) and live swarm (FIG. 3B) inhibit E. coli indicator lawn growth. TA−extract (FIG. 3C) and live swarm (FIG. 3D) does not inhibit indicatorlawn growth.

FIG. 4A is a graph demonstrating that production of antibiotic TAconfers a predation fitness advantage over a TA− mutant. FIG. 4B is agraph demonstrating E. coli predation by M. xanthus (DK1622).Prey/predator cells were mixed at a 10:1 ratio. TA^(r) #18 was derivedfrom E. coli imp-parent by selecting TA^(r). FIG. 4C provides a graphshowing predation of E. coli by a myxobacterium, Corrallococcuscoralloides c127, a producer of corallopyronin. PT agar was used,supplemented with 1 mM IPTG.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides methods for natural product optimization.More specifically, the instant invention provides new methods whichexploit the predatory behavior of certain microorganisms, includingmyxobacteria and other predatory gliding bacteria (e.g., lysobacter,cyctophaga, flexibacter, and pseudomonads). Optimized strains fornatural product (e.g., antibiotic) production are subsequently selectedin accordance with the methods provided herein.

The instant invention provides a new means to optimize antibioticproduction in predatory microorganisms such as myxobacteria. The vastmajority of antibiotics to date are derived from natural products (e.g.,aminoglycosides, macrolides, ketolides, tetracyclines, vanomycin,β-lactams (e.g., penicilliums, cephalosporins, monobactams), rifampin,fosfomycin, bacitracin, streptogramins, and daptomycin). The majorsynthetic exceptions are fluoroquinolones, sulfonamides, andoxazolidones. Natural products provide a rich source of chemicaldiversity and complexity (Clardy et al. (2006) Nat. Biotechnol.,24:1541-1550). Furthermore, natural product antibiotics are particularlyuseful because they evolved over time to have broad antibacterialactivity and generally act on more than one molecular target, whichgenerally makes resistance development difficult. In contrast, theseproperties are difficult to design into synthetic compounds. Further,natural products are complex structures that make chemical synthesisdifficult. In addition, the major secondary metabolite producers, suchas the Actinomycetes (which are not predatory organisms), have alreadybeen heavily mined by the pharmaceutical industry resulting in very fewnew structures being found. The instant invention overcomes the aboveshortcomings with previous natural product antibiotics.

Although myxobacteria are a prolific source of secondary metabolites(Reichenbach, H. (2001) J. Ind. Microbiol. Biotechnol., 27:149-156),including over 20 basic antibiotic (antibacterial and antifungal)structures, they have largely been overlooked for new therapeutics. Todate, 100 basic structures and 500 structural variants have beencharacterized (Bode et al. (2006) J. Ind. Microbiol. Biotechnol.,33:577-588). These NPs have been found with relatively small screeningefforts (Reichenbach, H. (2001) J. Ind. Microbiol. Biotechnol.,27:149-156; Reichenbach et al. (1993) Biotechnol. Adv., 11:219-277).Most of these natural products represent novel structures and elicitinteresting biological responses. For instance, about 20 of the basicstructures are antibiotics and many of these compounds representexcellent leads for optimization. For example, aside from TA,myxopyronin/corallopyronin offer promise as bacterial RNA polymeraseinhibitors (Mukhopadhyay et al. (2008) Cell 135:295-307). Anotherpromising myxobacterial NP are the epothilones, which inhibitmicrotubule depolymerization similar to paclitaxel (Donovan et al.(2008) Oncology 22:408-416).

Preliminary data is provided herein which shows that predatorymicroorganisms, such as myxobacteria, produce antibiotics to kill andeat prey organisms. The methods of the instant invention describes howgenetic selection can be used to isolate strains that overproducenatural productions, produce different ratios of existing analog naturalproducts, make new analog natural products, and/or turn on crypticbiosynthetic pathways (i.e., not normally expressed) to produce newantibiotics. These methods are superior to the laborious screens forstrain optimization historically used in industry. Indeed, enormousnumbers, e.g. about 10¹⁰ to 10¹² or greater, of cells can be processedfor rare mutants that exhibit improved activity by the instant methods.Traditional industrial screening procedures can only process about 10³to 10⁵ mutants for optimal activity.

Myxobacteria are prolific producers of secondary metabolites in which˜500 novel basic and derivative structures have been found. A fewexamples of such compounds are provided in FIG. 1. Many of the secondarymetabolites and derivatives produced by myxobacteria have antimicrobialactivity. Myxobacteria are only exceeded by the actinomycetes and thegenus Bacillus in the number of known structures produced. Unlike thesebacteria, myxobacteria are predatory. They glide or swarm over solidsurfaces and feed as microbial “wolf packs.” Prey bacteria are digestedby the secretion of a battery of hydrolytic enzymes including proteases,lipases, nucleases, and cell wall degrading enzymes. While the role ofantibiotic production on predatory behavior had not previously beendefinitively established, data is provided herein which shows thatantibiotic production serves to neutralize or kill prey microbes whichthen allows the hydrolytic enzymes to digest prey cells. Indeed, thehydrolytic enzymes may not be active on live cells because they may notbe able to penetrate live cells, particularly the outer membrane ofGram-negative bacteria. Moreover, live cells can repair damage caused byhydrolytic enzymes. In contrast, small molecule antibiotics canpenetrate cellular membranes and block cellular metabolism andcompromise membrane integrity, thereby allowing the hydrolytic enzymesto degrade the cell. Based on the above, antibiotic production isessential under conditions where myxobacteria must kill and digest preycells to survive. By extension, conditions can be devised for theselection and isolation of mutant myxobacteria with improved antibioticproduction.

In accordance with one aspect of the instant invention, methods for drugoptimization are provided. In a particular embodiment, the methodscomprise maintaining a predatory microorganism under culture conditionswherein the only nutrient source is prey cells (e.g., bacterial cells)which cannot be utilized by the predatory microorganism. The predatorymicroorganism may be defective in predation (e.g., because the prey isnaturally resistant or prey is engineered or mutagenized to beresistant) and cannot grow or swarm when the prey cells are the solenutrient source. Similarly, under semi-rich growth conditions defects inmicrobial predation may allow prey cells to grow and consequentlyinhibit predatory growth by depletion of competing nutrients and/orphysically blocking predator cells from swarming. The methods mayfurther comprise mutagenizing (introducing a mutation into the cell) thepredatory microorganism and selecting those cells which demonstrateincreased predation of the prey cells (e.g., when presented as the onlynutrient source). The methods may optionally further compriseidentifying the natural products produced by the selected cells. In aparticular embodiment, the methods of the instant invention may comprisemultiple rounds of selection (e.g., multiple rounds of culturing withprey cells, optionally, with repeated mutagenization).

The predatory microorganism may be mutagenized by any means known in theart. Means for producing mutants are known to those skilled in the artand include, without limitation, the use of chemical and physicalmutagens as well as targeted nucleic acid mutagenesis (e.g., insertionalmutagenesis by introducing nucleic acid molecules (e.g., genes) into thecells). The mutants may also be spontaneous. Chemical mutagens include,without limitation, EMS (ethyl methane sulfonate; methanesulfonic acidethyl ester), N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU),procarbazine hydrochloride, chlorambucil, ICR191, cyclophosphamide,methyl methanesulfonate (MMS), diethyl sulfate, acrylamide monomer,triethylene melamine (TEM), melphalan, nitrogen mustard, vincristine,bromodeoxyuridine, dimethylnitrosamine,N-methyl-N′-nitro-Nitrosoguani-dine (MNNG), 7,12 dimethylbenzanthracene(DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, methanesulfonate, dimethyl sulfonate, O-6-methyl benzadine, ethidium bromide,tamoxifen, 8-hydroxyguanine, and derivatives and analogs thereof. In aparticular embodiment, the chemical mutagen is selected from the groupconsisting of EMS, ICR191, and MNNG. Physical mutagens include, withoutlimitation, the irradiation of cells (e.g., ultraviolet irradiation andionizing irradiation (gamma, beta, and alpha irradiation and x-raysirradiation). In a particular embodiment, the physical mutagen isultraviolet irradiation. With regard to the targeted nucleic acidmutagenesis, DNA can be inserted into the chromosome(s) of themicroorganism or present on autonomously replicating plamsids. New genes(i.e., from other sources) can be provided or native genes can beduplicated or provided in multiple copies. Moreover, cloned DNA on aplasmid can be randomly or rationally mutagenized and transformed cellscan be selected for improved antibiotic activity. For example, anantibiotic biosynthetic gene cluster (or individual genes) can bemutagenized by low fidelity PCR or chemical/UV mutagenesis creating alibrary of mutant clones, which can be subsequently transformed intopredatory bacteria, selecting for improved predation.

Predatory microorganisms (e.g., predatory gliding bacteria) which can beused in the instant invention include, without limitation, myxobacteria(e.g., Myxococcus xanthus, M. stipitatus, M. virescens and M. fulvus;myxobacteria within suborder Cystobacterinea (e.g., Archangium,Corallococcus, Cystobacter, Melittangium, and Stigmatella); andmyxobacteria within suborders Sorangineae and Nannocystineae (e.g.,Byssovorax, Chondromyces, Haliangium, Nannocystis, Polyangium, andSorangium)) and certain species within the genera Lysobacter,Flexibacter, Pseudomonas and Burkholderia, which are predators. Thepredatory microorganism may be also be genetically modified. Forexample, as described hereinbelow, foreign recombinant metabolitepathways may be introduced into the predatory microorganism foroptimization by predator-prey selection. Additionally, the predatorymicroorganism may be modified to comprise a knockout of at least onegene essential for the production of a natural product produced by thepredatory microorganism (e.g., a modification(s) and/or deletion(s)renders the naturally occurring gene nonfunctional). For example, asdescribed hereinbelow, the ta1 gene may be knocked out of the predatorymyxobacterium.

The cells selected for increased predatory action in the instant methodsmay, among other things, overproduce natural products, produce differentratios of natural products, and/or make new and novel derivatives ofnatural products. The methods of the instant invention may furthercomprise selecting for cells which have at least one of the abovedesired characteristics. For example, the cells selected for increasedpredation may be further screened to identify those which areoverproducers of a desired natural product or antibiotic.

Further, the predation selective pressure can be used to select thosecells which have an altered ratio of bioactive analogs. For example,some strains of myxobacteria are known make at least 30 closely relatedantibiotics called myxovirescins (also called antibiotic TA ormegovalicins). The analogs have differential activity against differentprey bacteria. Thus, when a particular prey is used for selection, themost potent derivative that is active on that particular prey can beselected for overexpression compared to other analogs/derivatives. Incontrast, when a different prey is used under selection conditions thebest/most potent derivative may be selected for overexpression with thatprey compared to other analogs/derivatives.

Additionally, the mutagenesis and selective pressure on predation may beused to induce the synthesis of new natural products, which can besubsequently screened for. Indeed, the mutagenesis/selective pressure onpredation can be used to induce expression of cryptic antibioticpathways. For example, the M. xanthus (strain DK1622) genome has beensequenced and found to encode at least 18 gene clusters for naturalproducts synthesis (Bode et al. (2006) J. Ind. Microbiol. Biotechnol.,33:577-588; Goldman et al. (2006) Proc. Natl. Acad. Sci.,103:15200-15205). However, only 5 secondary metabolites have been shownto be produced by this strain (Krug et al. (2008) Appl. Environ.Microbiol., 74:3058-3068). Thus, the other pathways are likely eithernot expressed or expressed at low levels during standard laboratorycultivation conditions. The selection process may result in theisolation of mutants that express those pathways, thereby facilitatingpredation.

In addition to increasing production or potency optimization, otherproperties of the natural product compound can be optimized. Suchproperties include, for example, the ability of a compound to penetratetarget cells or not to be effluxed from target cells. Here, prey cellscan be selected which have reduced permeability, such as Pseudomonasspecies or, alternatively, particular prey mutants with reducedpermeability, such as E. coli mucoid mutants. If efflux is a parameterfor optimization, then prey strains that over produce efflux pumps canbe chosen as prey, or bacterial species that are known to have highlevels of efflux pump activity can be chosen as prey. Physiochemicalproperties of the natural product, such as solubility, stability, orbinding properties to surfaces or proteins, can also be optimized. Tooptimize each parameter selective conditions can be devised accordingly.For example, if improved solubility is sought, then predation conditionsshould be devised under liquid culture conditions. Predatory mutantsthat have improved predation ability under this condition may produce anantibiotic with improved solubility. If reduced serum albumin binding issought, then protein albumin can be added to predation conditions as abinding competitor to select compound derivatives with reduced albuminbinding.

The prey cells can be any microorganism or organism capable of beingpreyed on by the predatory microorganism. In a particular embodiment,the prey cells are cells that have obtained resistance to a naturalproduct (e.g., antibiotic) produced by the predatory microorganism. Forexample, if a prey cell (e.g., an E. coli mutant) is resistant to theantibiotic myxopyronin (e.g., by generating a mutation in the targetprotein, RNA polymerase), then derivatives/analogs of myxopyronin may besynthesized by a mutagenized predatory microorganism which previouslysynthesized myxopyronin, such that the derivative/analog can now bindand inhibit that target. Notably, target and non-target prey cells canbe constructed to optimize natural products with specific activitiesagainst the target cells but not the non-target cells.

In another embodiment, the prey microorganism is a fungal cell. As such,the natural product selected for may be an antifungal agent. Forexample, myxobacteria can prey on fungal organisms, including pathogenicorganisms like Candida albicans, as a food source and are prolificproducers of antifungal compounds. The instant methods can be used to,for example, identify overproducers, producers of novel antifungalcompounds, and/or producers of different ratios of antifungalcompounds/derivatives/analogs.

In still another embodiment, the prey organism is a multicellularorganism such as, without limitation, parasitic worms such as nematodes.Certain predatory microorganisms are known to prey on and killmulticellular organism. Accordingly, the methods of the instantinvention can be used to identify can be used to select and optimizeanti-parasitic compound production.

In yet another embodiment, the prey cells may be eukaryotic or mammaliancells. Certain predatory microorganisms produce compounds that haveknown cytotoxic effect on mammalian cells. As a consequence thesecompounds have therapeutic potential for cancer treatment. For example,one compound produced by myxobacteria that has shown great promise inclinical trials to treat cancer is epothilone. This anti-cancer agentworks by interfering with microtubule polymerization and consequentlykills cancer cells. Accordingly, predatory microorganisms may bemutagenized and selected for their ability to predate on prey eukaryoticcells (e.g., mammalian or human cells or cancer cells) to allow for theselection of optimized production of natural products with eukaryoticcytotoxic effects, e.g., anti-cancer agents.

According to one aspect of the instant invention, the predationselection methods can also be employed for natural products that areheterologously produced in a predatory microorganism. The naturalproduct may be expressed in a foreign predatory host. For example, thenatural product epothilone has been expressed in a surrogate host bycloning the epothilone biosynthetic pathway from the endogenousproducer, Sorangium cellulosum, into a heterologous M. xanthus host. Thepredation selection methods described herein can then be performed onthe M. xanthus host comprising the epothilone biosynthetic pathway.

In accordance with another aspect of the invention, the compoundsidentified by the predation screening methods described herein may beused administered to a subject as a therapeutic (e.g., as anantibacterial, antifungal, anti-parasitic, anti-cancer agent). In aparticular embodiment, the compound may be used an antibacterial agentagainst periodontal disease (e.g., oral administration). The isolatednatural product may be contained within a composition comprising atleast one pharmaceutically acceptable carrier.

According to another aspect of the instant invention, predators may betested against prey cells in a fitness experiment. More specifically,predator cells and prey cells are mixed in culture or on plates and areallowed to compete for growth on rich or semi-rich media. Under richmedia conditions, both cell types can grow because rich nutrients areused. Under semi-rich growth conditions, defects in microbial predationallow prey cells to grow and consequently inhibit predatory growth bydepletion of competing nutrients and/or physically blocking predatorcells from swarming. A predator that produces more/better antibioticactivity (e.g., more potent, improved solubility, improved stability,improved cell penetration, and/or reduced efflux) would kill and/oroutcompete prey cells and other less competitive predator cells. Thismethod can also be used with non-predatory antibiotic producers (e.g.,actinomycetes). The fitness test may be used to further characterize thepredatory microorganisms identified by the screening methods describedherein (e.g., further select superior producers of antibiotic activity).In another embodiment, the fitness test may replace the step ofscreening for optimized natural product producers by using prey cells asthe sole nutrient source, in the methods described above. For example,the mutagenized predatory cells can be subjected to a fitness test withprey cells and those mutagenized predatory cells which kill and/oroutcompete prey cells better than other predatory cells can be enrichedor isolated as optimized natural product producers. Multiple rounds mayperformed.

DEFINITIONS

The term “natural products” generally refers to compounds isolated froman organism and which are preferably identified as having apharmacological activity.

The term “isolated” generally refers to material that is substantiallyor essentially free from components which accompanied the material priorto isolation. The term “isolated” is not meant to exclude artificial orsynthetic mixtures with other compounds or materials, or the presence ofimpurities that do not interfere with the desired activity, and that maybe present, for example, due to incomplete purification, or the additionof stabilizers.

A “carrier” refers to, for example, a diluent, adjuvant, excipient,auxilliary agent or vehicle with which an active agent of the presentinvention is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water or aqueous saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin.

“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal government or a state government. “Pharmaceuticallyacceptable” agents may be listed in the U.S. Pharmacopeia or othergenerally recognized pharmacopeia for use in animals, and moreparticularly in humans.

Terms that refer to being “anti” a type of target organism or cell type(e.g., antimicrobial, antiviral, antifungal, antibacterial,antiparasite, anticancer) refers to having any deleterious effects uponthose organisms or cells or their ability to cause symptoms in a host orpatient. Examples include, but are not limited to, the inhibition orprevention of growth or reproduction, killing of the organism or cells,and/or the inhibition of any metabolic activity of the target organism.The term “antibiotic” refers to any substance or compound that whencontacted with a living cell, organism, virus, or other entity capableof replication, results in a reduction of growth, viability, orpathogenicity of that entity. The term “anticancer agent” refers tocompounds capable of inhibiting the proliferation of cancer cells (tumorcells) or killing cancer cells.

As used herein, the term “mutagen” refers to a compound or process thatresults in the introduction of mutations in the genome of an organism.

The following examples are provided to illustrate various embodiments ofthe present invention. They are not intended to limit the invention inany way.

EXAMPLE 1

Antibiotic TA is also known as myxovirescin, megovalicin and M-230B(Miyashiro et al. (1988) J. Antibiot., 41:433-438; Onishi et al. (1984)J. Antibiot., 37:13-19; Gerth et al. (1982) J. Antibiot., 35:1454-1459;Rosenberg et al. (1973) Antimicrob. Agents Chemother., 4:507-513). Thisbroad-spectrum macrocyclic antibiotic is synthesized by a hybridpolyketide synthetase (PKS) and nonribosomal peptide synthetase (NRPS)(Simunovic et al. (2006) Chembiochem., 7:1206-1220). In DK1622, twovariants have been described (Simunovic et al. (2006) Chembiochem.,7:1206-1220; compound 1 is shown as FIG. 1A; compound 2 replaces the ═Oat position 20 with H,H). Other myxobacteria species, such as M.flavescens, produce six variants, while M. virescens Mx v48 makes atleast 30 variants (Simunovic et al. (2006) Chembiochem., 7:1206-1220;Gerth et al. (1982) J. Antibiot., 35:1454-1459). Some analogs havedifferential activity. For example, compound 2 has potent Pseudomonasaeruginosa activity (MIC ˜1 μg/ml; Miyashiro et al. (1988) J. Antibiot.,41:433-438). The production of NP mixtures may serve a selectiveadvantage, i.e. broader antibacterial spectrum.

Antibiotic TA is bactericidal and inhibits cell wall biosynthesis inGram-negative and Gram-positive bacteria (Zafriri et al. (1981)Antimicrob. Agents Chemother., 19:349-351; Mukhopadhyay et al. (2008)Cell 135:295-307; Donovan et al. (2008) Oncology 22:408-416; Goldman etal. (2006) Proc. Natl. Acad. Sci., 103:15200-15205; Krug et al. (2008)Appl. Environ. Microbiol., 74:3058-3068). 99% of growing E. coli cellsare killed within 30 minutes at 2 μg/ml of Antibiotic TA and activity isnot inhibited by sera (Rosenberg et al. (1996) J. Indust. Microbiol.,17:424-431). Antibiotic TA is not active on fungi, protozoa, oreukaryotic cells and has been shown to be safe in animals and humans(Manor et al. (1989) J. Clin. Periodontol., 16:621-624; Rosenberg et al.(1996) J. Indust. Microb., 17:424-431). However, further development ofTA has been hampered by difficult synthesis (˜40 steps) and lowfermentation yields (0.2 to 20 mg/L) (Content et al. (2003) Bioorg. Med.Chem. Lett., 13:321-325). Environmental isolates have been found tosynthesize over 30 TA structural variants that exhibit differentialantibacterial activity. Consequently, predation schemes might select forthe production of particular variants, depending on the prey bacteriaused.

Several lines of evidence indicate that TA inhibits bacterial cell wallbiosynthesis, as stated hereinabove. In addition to bactericidalactivity, TA treatment induces spheroplast production; a hallmark ofcell wall inhibitors (Rosenberg et al. (1973) Antimicrob. AgentsChemother., 4:507-513). Metabolic labeling experiments show that TAblocks peptidoglycan synthesis (Zafriri et al. (1981) Antimicrob. AgentsChemother., 19:349-351; Gerth et al. (1982) J. Antibiot., 35:1454-1459)by inhibiting lipid II polymerization. TA does not inhibit the finalcross-linking step catalyzed by transpeptidases (penicillin bindingproteins, PBPs), the target of β-lactams (Zafriri et al. (1981)Antimicrob. Agents Chemother., 19:349-351). These data indicate that TAmay block polymerization by inhibiting transglycosylase activity ofPBPs, perhaps similar to moenomycin (Lovering et al. (2007) Science315:1402-1405). Alternatively, TA may block translocation of lipid IIfrom the cytoplasm to the periplasmic space (Silver, L. L. (2003) Curr.Opin. Microbial., 6:431-438).

Antibiotic TA exhibits high adhesive properties toward biologic andnon-biological material (Rosenberg, et al. (1984) Biotechnology,796-799). For this reason, TA is a promising antibacterial agent to coatindwelling medical devices (such as catheters), and to prevent theformation of bacterial biofilms and the resulting, difficult-to-treatnosocomial infections (Simhi et al. (2000) FEMS Microbiol. Lett.,192:97-100). TA also strongly adheres to hard dental tissues with slowrelease times and extended antibacterial activity (Manor et al. (1985)J. Dent. Res., 64:1371-1373). These key characteristics, combined withgood antibacterial activity against periodontal pathogens make TA a goodantibiotic for dental applications. Indeed, human clinical trials showedthat TA was an effective therapeutic for periodontal diseases (Manor etal. (1989) J. Clin. Periodontol., 16:621-624; Eli et al. (1988) Refuat.Hashinayim., 6:14-15). The results found that in only two treatments,three indices were significantly improved (plaque, gingival, andbleeding) and were long-lasting.

The ta1 gene encodes a polyketide synthase that is required forbiosynthesis of the macrolide antibiotic TA (original isolate from TelAviv). The ta1 gene resides within an 83 kb gene cluster that encodes 21gene products involved in TA biosynthesis. The ta1 gene and operon arenot essential for growth in rich media (e.g., casitone).

A ta1 gene knockout construct may be made by PCR amplification of a 1.2kb internal gene fragment, which may be TOPO cloned into the pCR2.1plasmid. The resultant plasmid may be transformed into E. coli selectingkanamycin resistance. A validated clone may then be transformed into M.xanthus by again selecting Kan^(r). This plasmid cannot replicate in M.xanthus and thus transformant are derived from homologous integrationand gene disruption of the ta1 locus.

A defect in TA production may be determined in a bioassay, in whichsusceptible bacteria are streaked closely, but not touching, a mature M.xanthus colony. Alternatively, extracts may be purified from M. xanthuscultures and applied to a 5 mm diameter filter disk. This disk may beplaced on a lawn of bacteria to measure the zone of inhibition ofgrowth. Quantification of predation is scored by measuring swarm rates(plaques) on a lawn of susceptible prey as the only nutrient source.Notably, a single M. xanthus cell can give rise to a visible plaque (5-7days). Different TA susceptible bacteria may be used as prey.Alternatively, M. xanthus strains are mixed with prey cells and atvarious times the rate of prey killing is measured by collecting andserially diluting prey cells and counting cfu on agar plates.

As seen in FIG. 2C, the ta1 mutant shows a dramatic defect in predation.To quantify the effect of the ta− mutation on prey killing, a predationassay was done on ½ CTT agar plates (Wall et al. (1999) J. Bacteriol.,181:24-33). To help avoid the problem of non-specific killing of preycells caused by M. xanthus autolysis during sample preparation, the M.xanthus cells were pre-spotted on a agar surface 2 hours prior to preyplacement at a one-tenth ratio to prey cells (MG1655). FIG. 2C showsthat the TA− mutant exhibits a drastic defect (>7-logs) in predation.The initial dip in prey killing is attributed to residual M. xanthusautolysing and release of hydrolytic enzymes that inevitably happensduring M. xanthus sample preparation (Rosenberg et al. (1996) J. Indust.Microb., 17:424-431). These results clearly show that TA productionplays a key role in predation.

Thus, the ta1 mutant exhibits a predation defect. Experimentalconditions may be modified to accentuate a ta1 phenotype. Parameterswhich may be adjusted include prey, concentration of prey cells,buffer/salt, assay (liquid vs. agar plate), growth conditions of preycells, and quantification methods (swarm rates vs. viable colony formingunits). For example, M. xanthus does not utilize sugars as carbon/energysources, thus simply adding glucose can selectively allow prey cellgrowth. It is also important to note that the physiology of prey cells,e.g. growth vs. stasis, can effect predation.

To assess the impact of TA antibiotic product on predation, an isogenicwild type and a ta1 mutant may be grown on a lawn of susceptible preybacterial as the only source of nutrients. Quantification of predationability is scored by measuring and plotting swarm rates (plaques) of thetwo M. xanthus strains. If the myxobacteria are able to kill and digestprey cells, the initial inoculum will grow and will spread or swarm in aconcentric pattern through gliding motility over the agar surface.Bacteria that are known to be susceptible to antibiotic TA that will beused a prey cells include Escherichia coli, Klebsiella pneumonia,Bacillus subtilis, Enterobacter aerogenes, Micrococcus luteus,Staphylococcus aureus, and Enterococcus faecalis (see, e.g., FIGS. 2Aand 2B).

EXAMPLE 2

To test predation as a selection means for antibiotic optimization, theability of a ta1 mutant to revert back to the ta1⁺ allele can bemeasured. In the absence of Kan^(R) selection (plasmid retention)reversion occurs at a low frequency by homologous plasmid excision(reverse reaction of integration) resulting in a ta1⁺ allele. To allowexcision, the ta1 mutant may be grown for several days in rich casitonemedia in the absence of kanamycin. These cells may be harvested, washed,and applied to prey plates. Incubated plates may be visually inspectedfor swarming revertants. Putative revertants may be tested for Kan^(s)(plasmid excision) and confirmed by PCR. Ta1 revertants will be able togrow and swarm on prey cells. Moreover, the selective condition may besensitive to detect rare reversion events. The observance of revertantsdemonstrates the ability to use predation for antibiotic optimization.Notably, a certain threshold of revertant cells may be needed to bepresent to allow a pack of cells to kill, digest, and swarm over a preylawn. To assess this parameter, wild type M. xanthus cells may be mixedat different ratios with ta1 mutants to determine assay sensitivity.Other experimental parameters such as buffers, salts, prey species, andconditions may need refined, as stated hereinabove.

EXAMPLE 3

The biosynthetic pathway for antibiotic TA production is encoded within21 ORFs that span an 83 kb region (Simunovic et al. (2006) Chembiochem.,7:1206-1220). To elucidate a possible role of TA in predation, the genethat encodes a major megasynthase, ta-1, was inactivated by homologousrecombination with a suicide vector. The TA1 megasynthase (27 kb gene;978 kDa protein) is essential for TA biosynthesis (Paitan et al. (1999)J. Mol. Biol., 286:465-474). The ta1 gene knockout was constructed byPCR amplification of a 1.2 kb internal gene fragment (size allowsefficient recombination) and TOPO cloned into pCR2.1 (Invitrogen;Carlsbad, Calif.). A validated clone was then electroporated into M.xanthus and selected for homologous integration (Kan^(r)). A validatedrecombinant was then tested for antibiotic TA production with abioassay. Here, an extract from the TA− mutant (ta1::kan) was preparedand activity compared to an extract prepared in parallel from WT cells.As shown, the crude extract prepared from the TA− mutant lacksantibiotic activity compared to WT (FIGS. 3A and 3C). Next, a live TA−mutant swarm was tested for antibacterial activity against an indicatorlawn of E. coli cells. As shown in FIG. 3D, the TA− mutant swarm touchesthe E. coli lawn and does not form a zone of inhibition around the swarmedge. In contrast, the parental WT strain inhibits E. coli growth byforming a ˜3 mm clear zone around the swarm edge (FIG. 3B). Theseresults show that antibiotic TA plays an important role in killingbacteria.

EXAMPLE 4

It was then tested if antibiotic TA confers a selective fitnessadvantage over a strain that does not produce TA. In this experiment WT(DK1622) cells were mixed with the TA− mutant at a 1:1 ratio and spottedon a thick lawn of E. coli (MG1655) seeded on a TPM starvation agarplate (Wall et al. (1999) J. Bacteriol., 181:24-33). Under theseconditions the M. xanthus cells can only grow by killing and consumingprey cells. After a ˜10 day incubation the M. xanthus swarm nearlyconsumed the entire prey lawn. At this time the outer swarm edge wascollected with a sterile wood stick. The ratio of DK1622 to TA−(ta1::kan) cells was then determined by serial dilutions and replicaplating on CTT plates “with” and “without” kanamycin. Kan^(s) colonieswere scored as DK1622 and Kan^(r) colonies were scored as TA− cells. Inparallel the harvested M. xanthus mixture was also transferred to afresh TPM agar plate seeded with a naive lawn of E. coli. This cycle wasrepeated a total of 4 times (˜40 days). The relative ratios of DK1622(WT) to the TA− mutant were then plotted (FIG. 4A). As illustrated, theTA− mutant was drastically less fit than the isogenic parent (WT) andwas depleted from the M. xanthus population to undetectable levels(>5-logs). In a control experiment the TA− mutant and WT cell mixturewere treated identically, except the predation experiment was done on ½CTT (nutrient agar, thus predation not required for growth). In thiscontrol experiment, after the 4^(th) transfer there was no loss offitness for the TA− mutant. This result demonstrates that the ta1::kanmutation is stable and that the mutant is competent at swarming and isequally fit when nutrients are available. Importantly, these experimentsdemonstrate that TA production confers a selective fitness advantagewhen microbial predation is required for growth, supporting theunderlying thesis that microbial predation serves as a means to selectoptimized TA producing strains.

Antibiotic resistance by prey cells confers a corresponding resistanceto predation. In reciprocal experiments, prey mutants that are resistantto antibiotic TA were isolated to test if they become correspondinglyresistant to predation by the producer strain. Here, an E. coliimp-permeability mutant was mutagenized and resistant colonies wereselected on agar plates seeded with a TA extract. One TA^(r) mutant(#18) was further tested. In a MIC assay mutant #18 elicited a 64-foldincrease in resistance toward the TA extract as compared to the parentalstrain. Next, a predation assay was conducted with mutant #18 and itsparental strain against DK1622. As FIG. 4B illustrates, the TA^(r) #18mutant exhibits a corresponding and dramatic increase in resistancetoward predation. At times there is >8-log difference in killing betweenthe TA^(r) #18 mutant and the parent E. coli strain. These results againsupport the conclusion that antibiotic TA serves as a central weapon tokill prey bacteria.

EXAMPLE 5

Notably, the molecular target for TA is not known and the molecularmechanism for TA^(r) has not been elucidated. To unequivocally show adirect relationship between antibiotic resistance and predationresistance a better defined system was sought. One such myxobacteriasystem is for the NPs myxopyronin, corallopyronin and ripostatin. Thesethree NP antibiotics have recently been shown by x-ray crystallographyto bind to the “switch region” of bacterial RNA polymerase (Mukhopadhyayet al. (2008) Cell 135:295-307). In these studies the investigators alsoisolated RNAP mutations that conferred resistance to these antibiotics.The rpoB mutation contains a single mutation that results in a Val→Phesubstitution (position 1275). Relative to wild type cells, this mutationincreases antibiotic MIC values >32-fold Myx, >8-fold Cor and >16-foldRip. It was then tested if the RpoB-V1275F mutant was also resistant topredation by the myxobacterial producer strain for corallopyronin,Corallococcus coralloides c127 (Irschik et al. (1985) J. Antibiot.(Tokyo), 38:145-152). In these experiments the rpoB allele is present ona plasmid that is under lac control; consequently predation was done inthe presence of inducer (IPTG). E. coli predation was compared to anisogenic strain that has the wild type rpoB gene on a plasmid. As seenin FIG. 4C, the RpoB-V1275F mutant exhibits strong resistance towardspredation. At times there was an 8-log difference in predation comparedto the parental strain. Importantly, the only difference between theseE. coli prey strains is a single base-pair change in rpoB. These resultsare consistent and support the above results with mutant #18, namelythat resistance to antibiotics made by a producer strain confers acorresponding resistance to predation by that producer strain. Takentogether, the data indicates that antibiotic TA plays a key role inkilling prey and in predation fitness. By extension these resultsindicate that predation can be exploited as a means to select optimizedantibiotic TA producer strains.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

What is claimed is:
 1. A method for screening for a natural productproducing strain of a predatory microorganism, said method comprises: a)obtaining a predatory microorganism having a mutation that prevents themicroorganism from preying on prey cells; b) culturing the predatorymicroorganism of step a) under conditions wherein the predatorymicroorganism must compete with prey cells for growth, wherein saidpredatory microorganism cannot prey on said prey cells; c) isolating amutant predatory microorganism which grows under the culture conditionsof step b); and d) screening the isolated mutant predatory microorganismof step c) for altered natural product production compared to thepredatory microorganism of step a), wherein the isolated mutantpredatory microorganism of step d) is said natural product producingstrain.
 2. The method of claim 1, further comprising the step ofmutagenizing said predatory microorganism of step a).
 3. The method ofclaim 2, wherein said predatory microorganism is mutagenized by achemical mutagen or a physical mutagen.
 4. The method of claim 1,wherein said prey cell is resistant to the natural products produced bythe predatory microorganism of step a).
 5. The method of claim 1,wherein said prey cell is selected from the group consisting ofbacteria, fungus, parasite, mammalian cell, and cancer cell.
 6. Themethod of claim 5, wherein said prey is a bacteria.
 7. The method ofclaim 6, wherein said bacteria is selected from the group consisting ofEscherichia coli, Klebsiella pneumonia, Bacillus subtilis, Enterobacteraerogenes, Micrococcus luteus, Staphylococcus aureus, and Enterococcusfaecalis.
 8. The method of claim 7, wherein said bacteria is Escherichiacoli.
 9. The method of claim 1, wherein said predatory microorganism isa myxobacterium.
 10. The method of claim 9, wherein said mutation thatprevents the myxobacteria from preying on prey cells makes saidmyxobacteria unable to synthesize myxovirescin.
 11. The method of claim10, wherein said mutation is a ta1 knockout.
 12. The method of claim 9,wherein said myxobacterium is Myxococcus xanthus.
 13. The method ofclaim 1, further comprising isolating the natural product produced bythe isolated mutant predatory microorganism of step d).
 14. The methodof claim 1, wherein said natural product is selected from the groupconsisting of an antimicrobial compound, an antibiotic compound, anantifungal compound, an anticancer compound, and an antiparasiticcompound.
 15. The method of claim 1, wherein steps b) and c) arerepeated at least once.
 16. The method of claim 15, wherein the isolatedmutant predatory microorganism of step d) is mutagenized prior torepeating steps b) and c).
 17. The method of claim 1, wherein saidnatural product is a myxovirescin.
 18. The method of claim 1, whereinsaid predatory microorganism is Myxococcus xanthus, said prey isEscherichia coli, and said natural product is a myxovirescin.
 19. Themethod of claim 1, wherein said altered natural product production isselected from the group consisting of overproduction of a naturalproduct, production of different ratios of natural products, andproduction of natural products that were not produced by the predatorymicroorganism of step a).